The isolation of human stem cells offers the promise of a remarkable array of novel therapeutics. Biologic therapies derived from such cells through tissue regeneration and repairs as well as through targeted delivery of genetic material are expected to be effective in the treatment of a wide range of medical conditions. Efforts to analyze and assess the safety of using human stem cells in the clinical setting are vitally important to this endeavor.
Embryonic stem (ES) cells are the special kind of cells that can both duplicate themselves (self renew) and produce differentiated functionally specialized cell types. These stem cells are capable of becoming almost all of the specialized cells of the body and thus, may have the potential to generate replacement cells for a broad array of tissues and organs such as heart, pancreas, nervous tissue, muscle, cartilage and the like.
Stem cells have the capacity to divide and proliferate indefinitely in culture. Scientists use these two properties of stem cells to produce seemingly limitless supplies of most human cell types from stem cells, paving the way for the treatment of diseases by cell replacement. In fact, cell therapy has the potential to treat any disease that is associated with cell dysfunction or damage including stroke, diabetes, heart attack, spinal cord injury, cancer and AIDS. The potential of manipulation of stem cells to repair or replace diseased or damaged tissue has generated a great deal of excitement in the scientific, medical and biotechnology investment communities.
ES cells from various mammalian embryos have been successfully grown in the laboratory. Evans and Kaufman (1981) and Martin (1981) showed that it is possible to derive permanent lines of embryonic cells directly from mouse blastocysts. Thomson et al., (1995 and 1996) successfully derived permanent cell lines from rhesus and marmoset monkeys. Pluripotent cell lines have also been derived from pre-implantation embryos of several domestic and laboratory animal species such as bovines (Evans et al., 1990) Porcine (Evans et al., 1990, Notarianni et al., 1990), Sheep and goat (Meinecke-Tillmann and Meinecke, 1996, Notarianni et al., 1991), rabbit (Giles et al., 1993, Graves et al., 1993) Mink (Sukoyan et al., 1992) rat (Iannaccona et al., 1994) and Hamster (Doetschman et al., 1988). Recently, Thomson et al (1998) and Reubinoff et al (2000) have reported the derivation of human ES cell lines. These human ES cells resemble the rhesus monkey ES cell lines.
ES cells are found in the ICM of the human blastocyst, an early stage of the developing embryo lasting from the 4th to 7th day after fertilization. The blastocyst is the stage of embryonic development prior to implantation that contains two types of cells viz.    1. Trophectoderm: outer layer which gives extra embryonic membranes.    2. Inner cell mass (ICM): which forms the embryo proper.
In normal embryonic development, ES cells disappear after the 7th day and begin to form the three embryonic tissue layers. ES cells extracted from the ICM during the blastocyst stage, however, can be cultured in the laboratory and under the right conditions proliferate indefinitely. ES cells growing in this undifferentiated state retain the potential to differentiate into cells of all three embryonic tissue layers. Ultimately, the cells of the inner cell mass give rise to all the embryonic tissues. It is at this stage of embryogenesis, near the end of first week of development, that ES cells can be derived from the ICM of the blastocyst.
The ability to isolate ES cells from blastocysts and grow them in culture seems to depend in large part on the integrity and condition of the blastocyst from which the cells are derived. In short, the blastocyst that is large and has distinct inner cell mass tend to yield ES cells most efficiently. Several methods have been used for isolation of inner cell mass (ICM) for the establishment of embryonic stem cell lines. Most common methods are as follows:
1. Natural Hatching of the Blastocyst:
In this procedure blastocyst is allowed to hatch naturally after plating on the feeder layer. The hatching of the blastocyst usually takes place on day 6. The inner cell mass (ICM) of the hatched blastocyst develops an outgrowth. This outgrowth is removed mechanically and is subsequently grown for establishing embryonic stem cell lines. However, this procedure has few disadvantages. Firstly, Trophectoderm cells proliferate very fast in the given culture conditions and many a times, suppress the outgrowth of inner cell mass. Secondly, while removing the outgrowth of the inner cell mass mechanically, there is a chance of isolating trophectoderm cells. Thirdly, the percentage of blastocysts hatching spontaneously in humans is very low.
2. Microsurgery:
Another method of isolation of inner cell mass is mechanical aspiration called microsurgery. In this process, the blastocyst is held by the holding pipette using micromanipulator system and positioned in such a way that the inner cells mass (ICM) is at 9 O'Clock position. The inner cell mass (ICM) is aspirated using a biopsy needle which is beveled shape and is inserted into the blastocoel cavity. This procedure too is disadvantageous as the possibility to isolate the complete inner cell mass is low and many a time cells get disintegrated. It is a very tedious procedure and may cause severe damage to the embryo. The operation at the cellular level requires tools with micrometer precision, thereby minimizing damage and contamination.
3. Immunosurgery:
This is a commonly used procedure to isolate inner cell mass (ICM). The inner cell mass (ICM) is isolated by complement mediated lysis. In this procedure, the blastocyst is exposed either to acid tyrode solution or pronase enzyme solution in order to remove the zona pellucida (shell) of blastocyst. The zona free embryo is then exposed to human surface antibody for about 30 min to one hour. This is followed by exposure of embryos to guinea pig complement in order to lyse the trophectoderm. This complement mediated lysed trophectoderm cells are removed from inner cell mass (ICM) by repeated mechanical pipetting with a finely drawn Pasteur pipette. All the embryonic stem cell lines reported currently in the literature have been derived by this method. However, this method has several disadvantages. Firstly, the embryo is exposed for a long time to acid tyrode or pronase causing deleterious effects on embryo, thereby reducing the viability of embryos proper. Secondly, it is time consuming procedure as it takes about 1.5 to 2.0 hours. (Narula et al.,1996). Thirdly, the yield of inner cell mass (ICM) per blastocyst is low. Fourthly, critical storage conditions are required for antibody and complement used in the process. Lastly, it involves the risk of transmission of virus and bacteria of animal origin to humans, as animal derived antibodies and complement are used in the process. In this process, two animal sera are used. One is rabbit antihuman antiserum and the other is guinea pig complement sera.
The human cell lines studied to date are mainly derived by using a method of immunosurgery, where animal based antisera and complement was used.
Other possible disadvantages of the existing cell lines are as follows:    1. Use of feeder cells for culturing the human embryonic stem cell (hESC ) lines produces mixed cell population that require the Embryonic stem cells (ESC) to be separated from feeder cell components and this impairs scale up.    2. Embryonic stem cells (ESC) get contaminated by transcripts from feeder cells and cannot be used on a commercial scale. It can be used only for research purposes.
Geron established a procedure where human Embryonic Stem Cell (hESC) line was cultured in the absence of feeder cells (XU et.al 2001). The hESC were cultured on an in an undifferentiated state. The hESC contained no xenogenic components of cancerous origin from other cells in the culture. Also, the production of hESC cells and their derivatives were more suited for commercial production. In this process, there was no need to produce feeder cells on an ongoing basis to support the culture, and the passaging of cells could be done mechanically. However, the main disadvantage of this procedure is that the inner cell mass (ICM) is isolated by immunosurgery method, wherein the initial derivation of Embryonic Stem Cells is carried out using feeder layer containing xenogenic components. This raises the issue of possible contamination with animal origin viruses and bacteria.
In order to simplify the procedure of inner cell mass isolation and to make it safe, the scientists of the present invention have come out with a novel method of isolation of the inner cell mass using a non-contact laser, wherein, the use of animal based antisera and complement have been eliminated.
Use of Laser Technique in Assisted Reproduction:
With the advent of assisted reproductive technologies (ART), several methods have been used for improving fertilization, facilitating blastocyst hatching (Cohen et al, 1990) and performing blastomere biopsy (Tarin and Handyside, 1993). Commonly used methods are chemical (Gordon and Talansky 1986), mechanical (Depypere et al., 1988) and laser (Feichtinger et al., 1992) so as to produce holes in the zona pellucida (Gordon, 1988). Recently, an infrared 1.48 μm diode laser beem focused through a microscope objective was shown to allow rapid, easy and non-touch microdrilling of mouse and human zona pellucida and high degree of accuracy was maintained under conventional culture conditions (Rink et al., 1994). The drilling effect was shown due to a highly localized heat-dependent disruption of the zona pellucida glycoprotein matrix (Rink et al., 1996). Contrary to the detrimental effect on compacted mouse embryos induced by the 308 nm xenon-chlorine excimer laser (Neev et al., 1993), the drilling process in the infrared region did not affect embryo survival in mice (Germond et al., 1995) or in humans (Antinori et al., 1994).
Currently, lasers are being investigated as a tool to aid fertilization and in assisted hatching. Recent reports show that use of 1.48 μm diode laser for microdrilling mouse zona pellucida is highly safe and does not affect neuro-anatomical and neurochemical properties in mice and also improves fertilization (Germond et al., 1996). Obruca and colleagues first reported the success of laser-assisted hatching in human IVF in 1994. In this study, a 20- to 30-micron hole made in the zona pellucida (ZP) when the Patients with previous IVF failures from two separate centers were included in this study. There was a higher implantation rate per embryo in the laser-assisted hatching group (14.4%) versus the control group (6%). Pregnancy rates per transfer were also improved (40% versus 16.2%).
In a separate study, Er:YAG laser was used to thin the ZP of embryos derived from patients undergoing repeated IVF. Using a laser for thinning the ZP, embryologists are able to achieve accurate reduction of the ZP by 50%, which is very difficult with acidic Tyrode's solution. Presence of Acid Tyrode's solution near the embryo may also be detrimental. The rate of clinical pregnancies in the laser-hatched group was 42.7%, as compared to 23.1% in the control unhatched group. Since this data looked promising, the indication of laser-assisted hatching was extended. Women undergoing IVF for the first time yielded 39.6% clinical pregnancy rate in the laser-treated group versus a 19% rate in the control unhatched group (Parikh et al 1996).
During the last decade there has been ongoing research on the isolation of inner cell mass (ICM), as it is useful in establishing embryonic stem cell lines which in turn have the ability to develop into most of the specialized cells in the human body including blood, skin, muscle and nerve cells. They also have the capacity to divide and proliferate indefinitely in culture.
The present invention involves the isolation of inner cell mass (ICM), using laser ablation technique without undergoing the cumbersome procedure of immunosurgery. Hence, in the present invention, the use of animal derived antibodies or sera are eliminated and the procedure is safe, simple, rapid, and commercially viable.
The present invention, obviates the shortcomings associated with the conventional methods of isolation of inner cell mass (ICM). The inner cell mass (ICM) isolated by the present invention is found to be intact without causing any destruction or damage to the cells. The present invention thus provides a quick reliable and non-invasive method for isolation of inner cell mass (ICM). It also completely ruptures the trophectoderm thereby minimizing the contamination of inner cell mass (ICM), thus ensuring the purity of inner cell mass (ICM).